Quantification of algal viruses in marine samples

4 Quantification of Algal Viruses in Marine Samples StevenWWilhelm and Leo Poorvin Department of Microbiology, The University of Tennessee,Knoxville,TN 3 7996, USA

CONTENTS Introduction and background Concentration of viruses in water samples by ultrafiltration Most probable number (MPN) assays Plaque assays Conclusions

~HI,~I,41,~HI, I N T R O D U C T I O N

AND

BACKGROUND

Phycoviruses (viruses that infect either cyanobacteria or eukaryotic algae) impart significant mortality on their hosts in aquatic environments. Microorganisms (both eukaryotic and prokaryotic) in marine systems are thought to be responsible for as m u c h as 50% of the photosynthetic carbon fixation on the planet (Field et al., 1988). It is therefore apparent that agents of mortality that act directly to reduce primary production in marine environments will alter carbon and energy flux through these systems (Wilhelm and Suttle, 1999; Fuhrman, 1999). This has, in part, led to the increased interest in the ecology of marine viruses that has occurred through the last decade. Studies concerning the distribution and activity of viruses at the c o m m u n i t y level c o m m o n l y rely on direct counts to monitor changes in the natural viral community. While this information is pertinent to m a n y studies, it does not address the issue of the infectivity of these viruses or the range of organisms that m a y be directly influenced by viral activity. Outside molecular techniques (see below), the identification and enumeration of phycoviruses requires the observation of interactions between virus and their hosts. It is therefore pertinent to m a n y studies to be able to quantify the a b u n d a n c e of infective viruses that m a y impart mortality on specific phytoplankton. However, as these measurements require that virus-host interactions be observed, it is necessary from the onset that the host p h y t o p l a n k t o n be cultivable. Therefore, the techniques highlighted in this paper require that the host organisms can be cultured in the lab in order to enumerate potential viruses. METHODS IN MICROBIOLOGY,VOI~UME30 ISBN (t 12 521530-4

Copyright © 2001 Academic Press I.td All rights of reproduction in any form reserved

The identification of viruses in the sea that infect specific cyanobacteria and bacteria is still in its relative infancy compared to studies on viruses infecting marine heterotrophic prokaryotes (Suttle, 1996). While the total abundance of virus-like particles ranges from 10~ to 10~ m l ' seawater (Wilhelm and Suttle, 1999), viruses infecting and lysing phytoplankton only represent a subset of this population. However, viruses infecting the marine Synechococcus spp. commonly occur at concentrations > 10~ml ' in coastal waters, and have estimated at concentrations as high as 2.5 × 10~ ml' (Suttle and Chan, 1993; 1994; Waterbury and Valois, 1993). Concentrations of viruses infecting eukaryotic phytoplankton can be equally as high; Cottrell and Suttle (1995) measured abundances of lytic viruses infecting Micromonas pusilla at > 10~ ml '. Viruses infecting other phytoplankton, including Aureococcus anophag~{frrens (Milligan and Cosper, 1994), Chrysochromulina spp. (Suttle and Chan, 1995), Emiliania huxleyi (Bratbak et al., 1993), Heterosigma akashiwo (Nagasaki and Yamaguchi, 1997; Lawrence et al., 2000) and Phaeocystis pouchetii (Jacobsen et al., 1996) have also been isolated from pelagic marine systems in recent years. In recent years it has also been demonstrated that infectious phycoviruses can also be isolated from marine sediments. In the Western Gulf of Mexico, Rodda et al. (1996) found cyanophages in concentrations ranging from 9.4 x 10* m l ' at the sediment/water interface of a 47 m water column, to 3.0 x 102 ml ~at 30 cm below the sediment surface. As the water over the sediment contained an order of magnitude less virus, this suggests that the vertical transport and subsequent burial of infectious cyanophage or infected cyanobacteria was occurring (as the production of cyanophage in the absence of light is unlikely). As molecular techniques for the enumeration of phycoviruses are currently under development, it remains premature to include them as protocols in this chapter. Using the polymerase chain reaction (PCR) and virus specific primers, Suttle and co-workers have been able to establish a baseline of information on the genetic diversity of one group of algal viruses, the Phycodnaviridae (Chen and Suttle, 1996; Short and Suttle, 1999). Recently they have been able to estimate the diversity of at least a portion of the Phycodnaviridae using degenerate primers for the segments of the DNA polymerase genes of these algal viruses and denaturing gradient gel electrophoresis. Similarly, Fuller et al. (1998) have described the genetic diversity of cyanophage isolates infecting Synechococcus spp. using PCR techniques targeted at the DNA region encoding a capsid assembly protein. However, as with studies involving viruses infecting eukaryotic phytoplankton, these results remain qualitative. The advent of new techniques (e.g. quantitative PCR, in situ PCR) will hopefully soon provide qualitative values for the distributions of these algal viruses. This review describes the current methods available for the enumeration of specific viruses infecting phytoplankton in aquatic environments. It represents a compilation of methods that have been employed for many years in classic virology and those that have been adapted for use by 'viral ecologists' working in natural systems. Two approaches, the plaque assay $4

and MPN assay, are described here which allow researchers to both e n u m e r a t e and isolate viruses that lytically infect marine photoautotrophs.

C O N C E N T R A T I O N OF VIRUSES IN W A T E R SAMPLES BY U L T R A F I L T R A T I O N Principle In m a n y situations the a b u n d a n c e of lyric phycoviruses in a natural water sample is too low to accurately quantify. In these cases, the use of ultrafiltration techniques m a y be required to increase the concentration of viruses in the sample. Ultrafiltration involves the removal of bacterial and algal c o m p o n e n t s (> 0.2 pro) of the microbial c o m m u n i t y followed by the concentration of the 'viral size fraction' (typically 30 kDa to 0.2 pm). Small scale (0.3-20 ml) ultrafiltrations can be carried out with a commercially available centrifugation systems ('spin-columns') such as Centriprep or Centriplus units (Millipore). While these often will w o r k well with laboratory virus-host systems, these sample sizes are often too small to properly examine environmental samples. In these cases, techniques such as tangential flow filtration or vortex flow filtration can be used to handle larger volumes (1-200 1). With these techniques, concentration of the viral particles is achieved by successive circulation of the sample across a 30 kDa m e m b r a n e surface. This allows the water to be removed from the sample (ultrafiltrate) while the viruses are concentrated into the retained volume. The resulting viral concentrate can then be used as the 'sample' to screen (as described below). In this protocol, we describe the use of the Amicon M12 ultrafiltration system, as this is the system currently in use in our laboratory (adapted from Chen et al., 1996). Similar systems are p r o v i d e d by other suppliers, and it is suggested that the reader consider these other alternatives prior to making any investment in a system.

Equipment and reagents • Submersible pump with pressure gauge. • Two containers for water samples (20-200 I each, depending on volume to be concentrated). • 142 mm diameter glass fiber filters (MSF GC50; nominal pore size, 1.2 IJm) with holder(s) and appropriate tubing (non-toxic). • 142 mm diameter, 0.2 lure nominal pore-size filters (polycarbonate or low protein binding) with holder(s) and appropriate tubing. • Amicon ProFlux M-12 ultrafiltration system with non-toxic tubing designed for use in peristaltic pumps (such as Masterflex from PharMed). • Millipore S IOY30 spiral wound membrane cartridges (30 kDa molecular weight cutoff). • Header kits for S I0 cartridges.

55

Application Collect the water sample (20-200 1) into one of the holding containers. Using the submersible p u m p , prefilter (at < 17kPa) the sample first through the 142 m m diameter glass fiber filters (MSF GC50; nominal poresize, 1.2 ]am). Two or more of these m a y be set up in parallel for larger sample volumes. Follow this by filtering the sample through a 0.2 l~m filter into the second container. These filters will remove large particulates, algae, bacteria, etc. but will allow most viruses to pass. T h r o u g h o u t these steps, subsamples of water should be collected so that the recovery efficiency of this process can be calculated (see below). After filtration, use an Amicon ProFlux M-12 ultrafiltration system to concentrate the filtrate containing the viruses. Set the M-12 u p for concentration mode, with a Millipore $10Y30 ($10) spiral w o u n d m e m b r a n e cartridge (30 kDa cutoff). This cartridge, with a total m e m b r a n e area of 0.93m 2, will allow water to pass through but retain virus particles. Connect tubing from the container with the filtrate into the p u m p inlet of the M-12. Standard operating procedures involve running the p u m p at 40 to 50% of the m a x i m u m speed, with 50 to 60 kPa of backpressure. From the p u m p , run the tubing to the inlet header of the S10 cartridge and from the outlet header back to the container holding the sample to return the retenate. Connect tubing from the permeate connector to remove the ultrafiltrate, which can be discarded. As the system runs, the permeate (without virus particles) is removed, thereby concentrating the viruses in the remaining retenate. With this setup, a v o l u m e of 200 1 of seawater can be concentrated to 500 ml in about 1 hour. Take care not to attempt to reduce the v o l u m e of the retentate below the s u m m e d v o l u m e of the cartridge and the tubing. Measure the final volume of viral concentrate so that the concentration factor (CF) can be estimated as follows:

CF = volume qf sample / volume of retentate If required, the retenate can be further concentrated to a smaller v o l u m e (100 to 200 ml) using a smaller system (e.g. an $1Y30 cartridge, Chen and Suttle, 1996). Alternatively, other low-cost methods are available to concentrate samples. Centrifuge-based concentrators (e.g. Centriprep, Centriplus) can be used to concentrate viruses in small volumes of sample. The recovery efficiency (a.k.a. concentration efficiency) of this process must be determined w h e n using viral concentrates to estimate the abundance of infectious phycoviruses. This can be most easily determined by direct counts of the total viral abundance pre- and post-concentration (see chapter by Noble for direct counting techniques). The recovery efficiency (RF, as a %) is determined as follows:

RF = 100 (A,,,,/A ,,,,) / CF where A , , is the abundance of virus particles in the viral concentrate, A ...... is the abundance of viruses in the original sample, and CF is the concen-

56

tration factor (determined above). Typical recovery efficiencies vary, but are generally greater than 50% and commonly approach 100% (Suttle et al., 1991; Wommack et al., 1995).

Troubleshooting A series of problems can occur when making and using viral concentrates. Most problems are associated with the concentration of the viruses. The problems include leakage from old tubing, loss of viruses during prefiltration, and incorrect estimates of the viral concentration factor. Establishing familiarity with the concentration equipment and procedure(s) is a sure cure for many of these problems. The other considerations to be made with ultrafiltration are problems associated with filter integrity and cleaning. Breakthrough of viruses in damaged filters can seriously hinder concentration efficiency. All manufacturers, however, provide instruction on testing the integrity of their filters. As filters are often costly, cleaning procedures are important and designed to maximize the life of the filter cartridge. In the case of the system described, we recirculate 0.1 M NaOH to remove residual organics from the filter after each concentrate is made. The NaOH is subsequently removed using ddH:O, dilute H~PO~ and then ddH~O. Again, all manufacturers provide information on the chemical compatibility of their filters, and this should be checked in each case. This is especially important as some sterilizing agents (hydrogen peroxide, bleach, strong NaOH) can damage certain membranes and thus compromise their integrity.

• , ~

MOST PROBABLE NUMBER (MPN)ASSAYS

Principle Assessment of lytic viral activity requires that the virus particle destroys a host cell. Using a dilution approach, we can estimate the abundance of viruses in a sample. This process is based on the theoretical assumption that a single infectious virus can destroy a population of sensitive host cells (given time). The MPN approach to quantifying infectious viruses involves the exposure of a series of log-based dilutions of the sample containing the viruses to a liquid culture of host cells. Given an appropriate range of dilution (crossing the range where the mean viruses per aliquot sample is approximately 1.0) then the abundance of infectious viruses can be estimated. While the individual MPN exposures are scored '_+', comparison of these scores to MPN tables (or analysis by computer software) allows for an estimate of infective units. Replication can be achieved in several ways, with many labs now commonly using multi-well plates to enhance this (see Alternative technique). Ultimately, the desired levels of sensitivity and accuracy will dictate the volumes, number of replicates and scale of cultures to be used.

57

Equipment and reagents •

Culture medium for marine phytoplankton. ESAW (Harrison et al., 1980)

• • •

and its derivations commonly work well as a general growth medium. Liquid culture of host organism in exponential growth phase. 7 ml glass culture tubes (13 × 100 mm) with polypropylene screw caps. Fluorometer with filter set for chlorophyll determinations (Ks, 420ox;

•

>6400m). 25 mm, 0.22 lum nominal pore-size low protein binding filters (e.g.

•

Durapores®). Filtration funnel and receiver or Swinnex ® filter holder (and 10 cm 3

• •

syringe) for 25 mm filters. Pipette ( I - 5 ml) and tips for liquid dispensing. Culture facilities for phytoplankton.

Alternative technique (requires the following materials) • 96-well microtiter plates with lids. • Multichannel pipette and tips. • Fluorescent plate reader with filters for chlorophyll (Xs, 4200x; > 640om).

Application

(using 7 ml culture

tubes)

Collect water samples for screening into sterile p o l y p r o p y l e n e or polycarbonate containers and maintain them at 4°C in the dark until they are screened. Screen samples as soon as possible. Prior to screening, filter 25 ml of sample through a 0.22 p m nominal pore-size filter to remove bacteria, algae, protists, etc. In some cases other pore-size filters can be used (see Troubleshooting). From this, make a set of serial dilutions (10fold dilutions with sterile culture medium) with the sample to provide a dilution range of I to 10 4of the sample. A d d 1 ml of each of these dilutions to the exponentially growing host cultures (below) to screen for lytic activity. To prepare hosts for screening, transfer an aliquot of the host to fresh culture medium. For example, transfer 50 ml of an exponentially growing batch culture into 450 ml of medium. Monitor growth in this culture so that cells can be used as soon as the exponential phase of growth begins. As exponential growth begins, transfer 5 ml to each of fifty-five 7 ml culture tubes, assuming that ten replicates (and five controls with no sample added) is the desired n u m b e r for the experiment, and that five dilutions are being used (Suttle, 1993). Gently mix the tubes, record the fluorescence and place the tubes in culture facilities. Remove the tubes daily (for u p to seven days) and repeat the measurement of fluorescence. Cultures not clearing in seven days are assumed to not contain virus. For each dilution, record the n u m b e r of tubes that have cleared and use this data to calculate the MPN for the concentration of viruses in the sample. The MPN can be determined from published values in tables

58

(Koch, 1981) or by using c o m p u t e r programs which can also provide confidence intervals and standard estimates of error (Hurley and Roscoe 1983). Sample results are s h o w n in Figure 4.1.

500

400

Cyanophage P6 ! Cyanophage P56 [ Control !

T

i i

TJ soo t~ 0

T

200

m

ii 100

1

2

3

Time (days) Figure 4.1. Typical chlorophyll fluorescence from the cyanobacteria SyJlechococcus sp. WH7803 in culture with and without added viruses. The addition of cyanophage P6 demonstrates tile typical clearing seen in tubes during MPN assays. Both the control and cyanophage P56 (which does not infect this Synechococcus) demonstrate no clearing, and this was consistent up to 7 days (not shown).

Alternative technique As described by Suttle and Chan (1993) and Bratbak et al. (1998), microtiter plates can be substituted for culture tubes to screen cultures that will grow in these systems. Repeat the process as above, but substitute a 96-well microtiter plate for the 7 m l culture tubes and adjust volumes of host (100 tJl) and virus (50 1_ll). Maintain cultures u n d e r standard growth conditions and screen them daily, either visually or with a fluorescence plate reader equipped to monitor chlorophyll fluorescence. When using microtiter plates, it is easy to expand the dilutions from 5 to 7 (or more) tenfold steps. However, in the microtiter assay less sample is screened, so the m i n i m u m detection limits (sensitivity) of the assay is decreased.

Troubleshooting One problem c o m m o n l y associated with the screening of natural samples is the breakthrough of u n w a n t e d organisms (e.g. bacteria and protozoans) through the filter into the sample to be screened. This problem is often difficult to diagnose until after the experiment has been carried out, but samples can be examined by epifluorescence microscopy to determine the $9

presence of u n w a n t e d organisms if this problem becomes of concern. Another problem that occurs is the destruction or removal of viruses in the sample during storage, often by bacteria or protozoan grazers. To avoid this problem, filter samples upon collection (as described) and store in the dark at 4°C until use. It should be pointed out that viral infectivity will decay, even under these conditions. However, in at least one case infective viruses have been found in these concentrates after storage for 7 years under the above conditions (Wilhelm and Suttle, unpublished data). Another problem to consider is the removal of infectious viral particles by filtration. Different size filters are commonly suggested in different protocols (e.g. 1.0 lain, Suttle, 1993; 0.45 lain, Garza and Suttle, 1998). While we have suggested the use of a 0.22 lain filter in this protocol, it should be considered that decreasing the pore-size of the prefilter increases the possibility of viral retention during that step. In any case, all filters will retain some degree of viruses during this step, so consistency in pore-size, filter matrix and technique (e.g. pressure) is critical in providing reproducible results. Growth in the controls must also be closely monitored. Should all or a subset of the controls not grow, then it is not possible to determine if clearing in the test cultures is due to viral activity or non-experimental effects. It is therefore important to have established the ability to consistently grow the host culture in the lab prior to attempting to quantify viral particles. Finally, while the choice of using culture tubes relative to microtiter plates is left to the investigator, we would like to point out that the use of microtiter plates decreases the detection limit for infective viruses in the samples. As described above, the tube method will increase the detection limit 20-fold relative to the microtiter method (as 20 times more sample is screened).

,,,,~

P L A Q U E ASSAYS

Principle Plaque assays are commonly used in bacteriophage studies in order to enumerate the abundance of infectious phage in a sample. These same techniques m a y be applied to the enumeration of phycoviruses. Plaque assays have the advantage over MPN assays of providing increased accuracy, but have the disadvantage of requiring that hosts cells must be culturable and provide a confluent lawn on agar solidified growth medium. The principle of the plaque assay is simple: it assumes that, within a complete lawn of organisms on a Petri plate, each individual virus will produce a clearing or 'plaque' where it has lysed the localized host cell population. The plaque assay also provides the added advantage of allowing individual plaques to be isolated directly from the plate, providing a clonal copy of each virus. Moreover, in some cases the presence of turbid plaques can be taken as an indication of a potential lysogenic virus (although significant testing is required to confirm this). 60

Equipment and reagents • Culture medium for marine phytoplankton. ESAW (Harrison et al., 1980) and its derivations commonly work well as a general growth medium. • Agar for the solidification of culture medium (e.g. BactoAgar from Difco). • Liquid culture of host organism in exponential growth phase. • Autoclave/microwave. • Temperature controlled water bath or dry block. • Microfuge. • Vortex mixer. • 25 ram, 0.22 pm nominal pore-size low protein binding filters (e.g.

Durapores).

m

< O

• Petri places (plastic, 15 × 100 mm). • I and 5 ml pipeccers and tips. • 1.5 ml microfuge tubes. • 13 × 100 mm disposable borosilicate glass cubes and rack(s). • Erlenmeyer culture flask (250 ml). • Culture facilities for algae.

Application Prior to screening cultures, plates for the establishment of confluent lawns must be created. Bottom agar for these plates is created by adding 1~ ( w / v ) agar to the appropriate culture m e d i u m and autoclave sterilizing the sample. After the m e d i u m is allowed to cool to 60°C, pour plates (15 to 20 ml) u n d e r sterile conditions and allow them to solidify. It is important that plates are only p o u r e d to = 50% capacity. Once dried, invert the plates and store them as other cultures plates (4°C, dark). They are usually good for up to a week or more. Top agar is also required for plaque assays. To prepare it, add 0.6% ( w / v ) agar to 100 ml of growth m e d i u m in a 250 ml flask or media bottle. If sealed after sterilization, this can be stored at room temperature. When required, the agar is remelted in a microwave oven and 2.5 ml aliquots placed into three 7 ml disposable culture tubes per sample to be screened. Maintain these tubes at a temperature between 45 and 47°C in the water b a t h / h o t block until use. Fill three tubes with top agar to use as controls. To prepare water samples for screenings, filter 25 ml of sample through a 0.22 lJm pore-size filter to remove bacteria, algae, protists, etc. as these organisms m a y cause false plaques to form. It might be necessary with some samples to carry out a series of dilutions prior to undertaking the plaque assay, as it is desirable to have only 20-200 infectious viruses per aliquot. These dilutions can be carried out as described above, using sterile marine m e d i u m and a series of culture tubes. To begin the plaque assay, start cultures of phototrophs to allow for yields of around 10 r ml ' of exponentially growing cells. Harvest the cells by gentle centrifugation (3000-5000g) and then resuspend them to around 1if' cells ml ~. When working with heterotrophic bacteria this 61

4.1

¢-

concentration step is generally not required. Transfer cells (500 lad to three sterile microfuge tubes, and 500 lJl of sample to each tube, then close the tubes and mix by vortexing. For each set of experiments add 500 ~1 of sterile culture m e d i u m to hosts in microfuge tubes to act as a control. After samples are combined in microfuge tubes, a brief spin in a microfuge will remove any liquid from the interior lid of the tubes. Allow samples to sit so that the virus can adsorb to the host cells. Adsorption times for heterotrophic bacteria c o m m o n l y range from 5-25 minutes, but 30-45 minutes is sufficient w h e n the kinetics of adsorption are u n k n o w n (Suttle, 1993). After the adsorption period, mix the contents of each microfuge tube with a tube of top agar by vortexing. Quickly p o u r this mixture onto the bottom agar in the Petri plate, and 'swirl' the sample on a flat surface to evenly distribute the top agar mixture. After the plates dry and solidify (=60 minutes), invert them and m o v e them to appropriate culture facilities. Enumeration of plaques on the plates occurs once confluent host lawns have established (2-6 days). Individual plaques are e n u m e r a t e d and assumed to represent the presence of one lytic virus in the samples. For statistical relevance, it is desirable to enumerate plates from dilutions with plaque abundances ranging from 20-200 per plate. Once plaques are enumerated, the abundance of viruses infecting the host can be determined as follows: A = (p / d) x ( 1 / v ) where A is the abundance of infectious viruses (ml '), p is the n u m b e r of plaques on the plate, d the dilution factor for that plate and v the v o l u m e (ml) of sample a d d e d (as described above, 0.5). A comparison of the two assays (most probable n u m b e r and plaque) is given in Table 4.1.

Table 4.1 Comparison of the most probable number assay with the plaque assay for the enumeration of phycoviruses Method

Advantages

Disadvantages

MPN assay

• Flexible with culture requirements • Amenable to high replication • Does not require growth on solidified medium • Enumerates only infective viruses • Higher accuracy than MPN assay • Provides indication of potential lysogens • Provides for easy purification of viruses • Enumerates only infective viruses

• Less precision than plaque assay • Low probability of detecting lysogens

Plaque assay

62

• Must be cultured on solidified medium • Natural bacteria can cause plaqueqike clearings

Troubleshooting The most significant problem associated with variations in plaque assay results is inconsistency of technique. Ensuring that cells are in the same phase of growth in each experiment is vital to providing reproducible results. As well, culture conditions (including temperature) should be held constant to allow for an intercomparison of samples. To account for variation, positive controls consisting of samples with a k n o w n abundance of infectious viruses can be included in every experiment. However, it should be r e m e m b e r that stored samples of viruses slowly lose infectivity over time, so samples that are examined 6 m o n t h s apart may not be comparable using the same positive control. It should be stressed that the plaque assay m e t h o d is often difficult to use with eukaryotic plankton (the exception being strains of Chlorella spp.). As well, certain cyanobacteria will not grow on standard agar, as it usually contains too m a n y impurities. Better growth of cyanobacteria on agar plates can be achieved by first removing these impurities (Waterbury and Wiley, 1988).

CONCLUSIONS Two methods for the enumeration of infectious phycoviruses as well as a m e t h o d to increase the concentration of viruses in a water sample are discussed here. Each m e t h o d for viral enumeration has its particular advantages and disadvantages (Table 4.1). The choice of the particular m e t h o d will ultimately d e p e n d on the culturability of the host ceils in question.

References Bratbak, G., Egge, J. and Heldal, M. (1993). Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms. Mar. Ecol. Prog. Ser. 93, 39 48. Bratbak, G., Jacobsen, A., Heldal, M., Nagasaki, K. and Thingstad, E (1998). Virus production in Phaeocystis pouchetii and its relation to host cell growth and nutrition. Aquatic Microbial Ecol. 16, 1 9. Chen, E and Suttle, C. A. (1996). Amplification of DNA polymerase gene fragments from viruses infecting microalgae. Appl. EHvipx~n.Microbiol. 61, 1274-1278. Chen, E, Suttle, C. A. and Short, S. M. (1996). Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes. Appl. Environ. Microbiol. 62, 2869-2874. Cottrell, M. T. and Suttle, C. A. (1995). Dynamics of a lyric virus infecting the photosynthetic marine picoflagellate Micromonas pusilla. LiutHol. Oceanog. 40, 730 739. Field, C., Behrenfeld, M., Randerson, J. and Falkowski, P. (1988). Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281, 237-240. Fuhrman, J. A. (1999). Marine viruses and their biogeochemical and ecological effects. Nature 399, 541-548.

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Fuller, N. J., Wilson, W. H., Joint, 1. R. and Mann, N. H. (1998). Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCR-based detection and quantification techniques. Appl. Environ. Microbiol. 64, 2051-2060. Garza, D. R. and Suttle, C. A. (1998). The effect of cyanophages on the mortality of Synechococcus spp. and selection for UV resistant viral communities. Microbial Ecol. 36, 281-292. Harrison, P., Waters, R. and Taylor, E (1980). A broad spectrum artificial seawater m e d i u m for coastal and open ocean phytoplankton. J. Phycol. 16, 28-35. Hurley, M. and Roscoe, M. E. (1983). Automated statistical analysis of microbial enumeration by dilution series. J. Appl. Bacteriol. 55, 159-164. Jocobsen, A., Bratbak, G. and Heldal, M. (1996). Isolation and caracterization of a virus infecting Phaeocystis pouchetii (Prymnesiophyceae). J. Phycol. 32, 923 927. Koch, A. L. (1981). Growth measurement. In: Mamml of Methods for General Bacteriology (P. Gerhardt, Ed.), p. 179. American Society of Microbiologists, Washington, DC. Lawrence, J. E., Chan, A. M. and Suttle, C. A. (2000). A novel virus causes lysis of the toxic bloom-forming alga, Heterosigma akashiwo (Raphidophyceae). J. Phycol. (in press). Milligan, K. L. D. and Cosper, E. M. (1994). Isolation of virus capable of lysing the brown tide microalga, Aureococcus aJiophagt~fferens. Science 266, 805-807. Nagasaki, K. and Yamaguchi, M. (1997). Isolation of a virus infectious to the harmful bloom causing microalga Heterosi~ma akashiwo (Raphidophyceae). Aquatic Microbial Ecol. 13, 135 140. Rodda, K., Clark, L., IngaI1, E. and Suttle, C.A. (1996). Infective cyanophages persist in anoxic sediments on the continental shelf of the Gulf of Mexico. EOS Trans., Am. Geophys. UuioJl 76(3), OS 51 I-6. Short, S. M. and Suttle, C. A. (1999) Use of the polymerase chain reaction and denaturing gradient gel electrophoresis to study diversity in natural virus communities. Hydrobiolosqa 401, 19-32. Suttle, C. A. (1993) Enumeration and isolation of viruses. In: Handbook of Aquatic Microbial Ecolok,y (P. E Kemp, B. E Sherr, B. B. Sherr, and J. J. Cole, Eds), pp. 121-134. Lewis Publishers, Ann Arbor, MI. Suttle, C. A. (1996). Community structure: viruses. In: Manual of Environmental Microbiology (C. Hurst, G. Knudson, M. Mclnerney, L. Stezenbach and M. Walter, Eds), pp. 272-277. ASM Press, Washington DC. Suttle, C. A. and Chan, A. M. (1993). Marine cyanophages infecting oceanic and coastal strains of SyHechococcus - - abundance, morphology, cross-infectivity and growth characteristics. Mar. Ecol. ProS. Set. 92, 99-109. Suttle, C. A. and Chan, A. M. (1994). Dynamics and distribution of cyanophages and their effect on marine Synechococcus spp. Appl. Environ. Microbiol. 60, 3167-3174. Suttle, C. A. and Chan, A. M. (1995). Viruses infecting the marine Prymnesiophyte Chrysochromulina spp.: isolation, preliminary characterization and natural abundance. Mar. Ecol. Pro,~¢.Ser. 118, 275-282. Suttle, C. A., Chan, A. M. and Cottrell, M. T. (1991). Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine phytoplankton. Appl. E~lviron. Microbiol. 57, 721-726. Waterbury, J. B. and Valois, E W. (1993). Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl. Environ. Microbiol. 59, 3393-3399. Waterbury, J. B. and Willey, J. M. (1988). Isolation and growth of marine planktonic cyanobacteria. Meth. Enzymol. 167, 100-105.

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Wilhelm, S. W. and Suttle, C. A. (1999). Viruses and nutrient cycles in the sea. BioScience 49, 781-788. Wommack, K. E., Hill, R. T. and Colwell, R. R. (1995). A simple method for the concentration of viruses from natural water samples. J. Microbiol. Meth. 22, 57-67.

List of suppliers The following is a selection of companies. For m o s t products, alternative suppliers are available.

Fisher Scientific Worldwide 50 Fadenl Road Springfield NJ 07081-3193, USA Phone: 1 973 467- 6400 Fax: I 973 376-1546 Disposables, m e d i a reagents, vortexers, microfuges

Millipore 80 Ashby Road Bedford, M A 01730, USA Phone: (800) MILLIPORE Fax: 1 781 533-3110 Ultrafiltration systems, m e m b r a n e filters a n d holders

6S

Turner Designs, Inc. 845 W. Maude Avenue Sunnyvale CA 94086, USA Phone: 1 408 749-0994 Fax: 1 408 749-0998 http://www.turuerdesis,,ns.conl Fluorometers